Shear Stress Regulates Angiogenesis Of Endothelial Progenitor Cells Through The Pim1/eNOS Pathway

Objective:To investigate the effect of shear stress on the expression of Pim1 in human endothelial progenitor cells(EPCs),its effect on angiogenesis and possible signaling mechanisms.Methods:EPCs were isolated from fresh human umbilical cord blood by density gradient centrifugation combined with differential adherence.Cytological identification was performed by testing the ability to phagocytose Dil-acLDL and bind FITC-UEA-I.The parallel plate flow chamber and the turbulent pump system were used to load unidirectional laminar flow(UF,12dyn/cm~2)and turbulent flow(DF,0.5±4dyn/cm~2)shear stress on EPCs,respectively.Real-time PCR and Western Blot were used to detect the expression levels of Pim1 m RNA and protein,respectively.Western Blot was used to detect changes in eNOS,p S1177-eNOS,and p S633-eNOS levels.Angiogenesis experiments were performed on Matrigel matrix membranes.Results:EPCs can form clonal colonies.Cells in the central part tend to be round-shaped.The edges are mostly spindle-shaped.Single adherent cells are mostly spindle-shaped.Under fluorescence microscope,after phagocytosing Dil-acLDL and binding to FITC-UEA-I,EPCs showed red fluorescence and green fluorescence in the cytoplasm,respectively.The Pim1 protein in EPCs was mainly distributed in the nucleus and perinuclear nuclei.The loading of UF makes the cells long spindle-shaped,tending to be uniformly arranged,and the angiogenesis ability is significantly enhanced;DF makes the cells appear polygonal,the morphological differences between cells become larger,and the angiogenesis ability is weakened.The levels of Pim1 m RNA and protein,p S633-eNOS and p S1177-eNOS in EPCs increased under the action of UF.After incubation of Sim-4a,a specific inhibitor of Pim1,the protein and m RNA of Pim1 were inhibited,and the angiogenesis ability of EPCs decreased in parallel with the expression of Pim1.Under the action of UF,the up-regulation of Pim1 can be inhibited by SMI-4a,while the up-regulation of eNOS-ser633 phosphorylation level is not significantly affected by SMI-4a;under the action of DF,SMI-4a can phosphorylation levels of Pim1 and eNOS-ser633 Both are down.Conclusion:UF can up-regulate the expression of Pim1 in EPCs;Pim1 is a key molecule in EPCs in angiogenesis;different modes of shear stress regulate the phosphorylation of eNOS through Pim1,which affects the angiogenesis of EPCs.